Design, Synthesis, and Performance of NTA-modified Lipids as Templates for Histidine-tagged Protein Crystallization

Current methods of drug discovery and development typically involve either structure-based drug design approaches or high-throughput screening methods for lead identification. The structure-based discovery process relies heavily upon the labor-intensive determination of three-dimensional protein structures using X-ray diffraction (XRD) and NMR techniques. Although tens of thousands of soluble protein structures have been resolved at the atomic level using these methods, there are many drug targets, particularly integral membrane proteins, whose structures have not been elucidated. In the case of XRD, this limitation often arises from the lack of reliable and predictable methods for protein crystallization. The extensive number of proteins crystallized by the interfacial templating approach suggests that this method may offer great promise for structural biology applications, particularly for proteins that do not readily form X-ray quality crystals from bulk solution. This review highlights the various approaches that have been used for templating of protein crystallization at gas-liquid and solid-liquid interfaces. Special emphasis is placed on the synthesis of NTA- and IDA-conjugated lipids, their use in templating and concentrating proteins at lipid monolayer interfaces, and protein structure elucidations that have been facilitated by these metal-chelating lipids. Current approaches to control the nucleation and growth of protein crystals involving microfluidics, dip pen nanolithography, and non-covalent symmetry-based templating approaches are also discussed. Taken together, the progress in this field suggests that the interfacial templating approach to crystallization can develop into a powerful tool for high-throughput structural analysis by providing a universal, readily controllable method for crystallizing proteins.